The Effect of Gold Nano Particles with Different Sizes on Streptococcus Species

Statement of the Problem: Streptococcus mutans, Streptococcus sanguinis, and Streptococcus salivarius are most common etiologic bacteria for dental caries. Different sizes of gold nanoparticles may have different antibacterial effects on these species. Purpose: This study aimed to compare the antibacterial effect of chlorhexidine and three sizes of gold nano particles (25, 60, 90nm) against clinical and standard strains of Streptococcus mutans, Streptococcus sanguinis, and Streptococcus salivarius. Materials and Method: In this cross-sectional study, the specimens were collected from 75 children aged 3-5 years old. Antibacterial effect of chlorhexidine and three sizes of gold nano particles (25, 60, 90nm) were investigated by evaluating the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against three bacterial strains. Results: The MIC and MBC of gold nanoparticles with different sizes against Streptococcus mutans, Streptococcus sanguinis, and Streptococcus salivarius were statistically different. The MIC and MBC of smaller gold nano particles (25nm) were significantly lower (p<0.001) than larger ones. Patient-derived bacteria had significantly higher values of MIC and MBC in comparison to standard species (p<0.001). Conclusion: The results of this study confirmed the significant size-dependency of gold nano particles for antibacterial activity. As the size of gold nano particles decrease, the antibacterial properties enhance.


Introduction
Nano is derived from the Greek word which means 'dwarf'; nanotechnology is a science that deals with manipulation of matter at the atomic level [1][2].
Nanobiotechnology is a field of applying nano scale techniques and biomaterials for inventing new treatments, medications, drug delivery systems [3][4], enzyme immobilization, and DNA transfection [2]. This science has been developed greatly. The efficacy of nano-particles can be affected by their size [5][6].
Dental caries is the most common human infectious disease in which diverse pathogenic factors and microorganisms have been identified such as streptococcus mutans (S. mutans), streptococcus salivarius (S. salivarius) and streptococcus sanguinis (S. sanguinis), salivary related disorders and individual diet [7][8].
Different treatments or preventive protocols have been introduced for dental caries. For centuries, metals have been proposed as antibacterial agents. Silver, gold, zinc, platinum [4,7] are the most common metallic agents. The antibacterial properties of metals can be affected by their contact area; larger surface of metals nanoparticles may cause more potent interactions with other molecules, which have not yet been determined [7,9]. Recently gold nanoparticles (AuNPs) have been introduced as a novel platform for new applications including nanobiotechnology and nanobiomedicine.
Gold nanoparticles have convenient surface bio conjunction and noticeable Plasmon resonance optional properties. In addition, they have antimicrobial effect and cause bacterial membrane damage, toxicity and aggregation interference [10]. To the best of our knowledge, there were few studies about comparing different sizes of AuNPs. On the other hand, many evaluations have been confirmed the nanoparticles antibacterial effect.
Martínez-Castañón et al. [11] evaluated the antibacterial properties of silver nanoparticles (7, 29, 89nm) against Escherichia coli (E.coli) and S.aureus. Decreasing in nanoparticle size, the antibacterial activity increased in the mentioned study. Smaller silver nanoparticles can present greatest surface area, interact with bacteria in a broader surface and reach the nuclear contact more easily. Hernández-Sierra et al. [7] have assessed the effect of silver, zinc oxide and gold nanoparticles with average sizes of 25, 125, 80nm on S.mutans. They have confirmed the increase in contact surface by reduction of nanoparticles size.
A study has reported antibacterial effect of gold and silver nanoparticles against E.coli and Bacillus Calmette-Guerin [5]. In addition, this was confirmed for silver and AuNPs against E.coli and S.aureus [3]. According to these researches, we aimed to evaluate antibacterial effects of different sizes of AuNPs against dental biofilm bacteria (such as S.mutans, S.salivarius and S.sanguinis).

Materials and Method
In this study, 75 children aged 3-5 years old, referred to Shiraz Dental Faculty, were enrolled in this study during 6 months. The Ethics Committee of Shiraz University of Medical Sciences has been approved this study (IR.SUMS.REC.1395.S1017). This study has been conducted according to the Declaration of Helsinki (1975). One of the participant's parents signed the written consent form. Dental caries of children was assessed by using dental explorer and bitewing radiographs [12]. A total of 75 specimens from teeth plaque with dental caries were achieved by a sterile toothpick.
In addition, a sterile cotton swab was employed for collecting unstimulated saliva from sublingual region. The samples were inserted into separate 1.0-mL reduced transport fluid vials [13] and sent to the microbiologic cen-ter (located in Jahrom, Fars province, Iran) for processing and laboratory evaluations. The saliva and plaque samples were diluted and placed on MM10-sucrose agar [14].
The cultures were incubated anaerobically (85% N 2 , 10% CO 2 and 5% H 2 ) and S. sanguinis colonies were selected based on their firm, adherent, star-shaped colony morphology [15][16], also those colonies with spherical and gram-positive anaerobic bacteria that were catalase and oxidase negative were S.salivarius. Blast analysis was used for assessing the candidate primers sequences in the database (http://www.ncbi. nlm.nih.gov/GenBank).
The genomic DNA was extracted according to the  They were sub-cultured in 5% sheep's blood agar.
At first, five to six colonies from an overnight culture were diluted in brain heart infusion broth and were incubated in an aerobic environmental condition for 1-2 hours at 35°C to reach the concentration of 1.    Figure 5 and 6 respectively.

Discussion
The antibacterial properties of AuNPs were inversely si-   Agnihotri et al. [6] confirmed size-specific antibacterial efficacy of silver nanoparticles against Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus).
Silver nanoparticles smaller than 10 nm showed considerable enhancement in antimicrobial activity; moreover, the smallest size mediated the fast bactericidal activity.
Findings of another study about the effect of size and shape of silver nanoparticles were in consistent with previous researches. MIC of 7nm silver nanoparticles against S. aureus and E.coli were the lowest [2].
In accordance to these studies Zhou et al. [5] evaluated silver nanoparticles antimicrobial effect against both aerobic and anaerobic oral pathogen; confirming the size-dependency of antibacterial activity of nanoparticles, the reported MIC for 5 and 15nm silver nanoparticles against S.mutans and S.sanguinis were 50µg/ml. Although the precise antibacterial mechanism of nanoparticles is unclear, there are some theories, which explain their mechanism [24][25]. Nanoparticles can attach to the cell membrane and disturb the permeability of the outer membrane. Therefore, they can enter the inner layer of membrane and stop respiratory chain dehydrogenase [24][25], disassociate the respiratory chain and oxidative phosphorylation and disable protonmotive force via cytoplasmic membrane [26]. Diminishing the size of nanoparticles can lead to more surface area of interaction whit bacterial cell membrane and increasing gold ions release and better antibacterial properties [19].
Electrostatic attraction between bacterial cell membrane and nanoparticles, produce a tendency to enhance nanoparticles accumulation [27][28] on bacterial cell membrane, which can lead to high stress in bacterial membrane and penetration of nanoparticles to cytoplasm and finally cell lysis [5]. In theory, interaction of nanoparticles with thiol groups of bacterial proteins may affect the DNA replication [29].
Several researches have theorized two possible mechanisms of antibacterial activity including increase in reactive oxygen species (ROS) production (hydroxyl radicals and singlet oxygen) [30][31] and disruption of cellular function by accumulation of nanoparticles on bacterial cell wall, in the cytoplasm or periplasm region [32][33]. Some nanoparticles can affect the bacterial junction and expression of cytokine gene [34].
The effect of nanoparticles on bacterial respiration can be explained by more resistancy of anaerobic oral bacteria such as S.mutans, S.sanguinis, S.mitis, and Actinobacillus actinomycetemcomitans. For anaerobic bacteria, the release of nanoparticles may be blocked by insufficient air; hence, the difference in releasing ions of nanoparticles makes the diversities in antimicrobial potencies for aerobic and anaerobic bacteria [35].
Beside this item, the effect of nanoparticles on gram-negative and gram-positive species is different because of different width of their cellular wall [36].
Although there is a controversy about the relation of concentration of nanoparticles and antibacterial effect [5,35], all articles support the size -dependency in a similar manner; as the size of nanoparticles decrease, the antimicrobial effect increase [7,11,19,35].
In the current study, the antibacterial potency of three different sizes of AuNPs was evaluated on both clinically isolated and standard species. This can help evaluate the trend of resistancy in oral pathogens.
According to findings of this study, unfortunately there is an increasing and concerning trend of antimicrobial resistancy in human isolated microorganisms. Therefore, the need for introducing new antimicrobial agents is completely necessary.
AuNP has been selected in our study for evaluation because of its solubility in water and in culture media.
Using water as our solvent can eliminate the antibacteri- signed nano products such as dentifrices and mouthwashes can be recommended for future studies.

Conclusion
The results of this study confirmed the significant sizedependency of AuNPs for antibacterial activity. As the size of AuNPs decrease, the antibacterial properties enhance. The patient-isolated bacteria are more resistant to antibacterial effect of AuNPs.